The basis of some of the embodiments of the present disclosure is the trapping of target molecules by a recognition reagent (referred to as a reader molecule) tethered to tunneling electrodes, which may be referred to as recognition tunneling (or RT). In a series of earlier disclosures, WO2009/117522A2, WO2010/042514A1, WO2009/117517, WO2008/124706A2, and WO 2011/097171, each of which is incorporated herein by reference, systems and methods are disclosed where nucleic acid bases may be read by using the electron tunneling current signals generated as the nucleo-bases pass through a tunnel gap consisting of two electrodes functionalized with reader molecules. A demonstration of the ability of this system to read individual bases embedded in a polymer was given by Huang et al.1 
RT may be also used to read an amino acid sequence, leading to protein sequencing in a nanopore/orifice, as set out in WO 2013/116509, entitled “Systems, Apparatuses And Methods For Reading an Amino Acid Sequence” (“the '509 publication”). In the '509 publication, two methods are disclosed for sequencing proteins based on recognition tunneling. In one method, an enzyme coupled to a functionalized orifice or nanopore is used to feed amino acids into a tunnel junction as they are sequentially cleaved from the end of a protein chain. The second method feeds intact peptides through a nanopore where the amino acid sequence is read out as each amino acid residue passes through the tunnel junction. In some of embodiments of the second approach, the method is simpler and more straightforward, and may be configured to produce longer sequence reads.
The '509 publication also describes methods and systems for reading negatively or positively charged peptides using a pair of nanopores—one biased to attract the positive peptides, the other biased to attract the negative peptides, and methods and systems where neutral peptides are pulled through a nanopore by electro osmosis. A possible issue with this approach is that it requires a separate arrangement for each type of net peptide charge, positive, negative and neutral. Furthermore, the approaches yield no information about which end, N or C terminus, of the peptide enters the nanopore first. Finally, the electrical force on the peptide varies with the charge on the peptide, so a significant run of neutral residues or residues of opposite charge to the overall charge of the peptide result in no force (or even a reversed force) on segments of the peptide. In fact, significant forces may be required to pull the peptide through the nanopore in order to overcome the tendency of the peptide to fold.
Accordingly, in view of the above-noted issues, it would be desirable to develop a method to draw peptides of any charge through a nanopore and to do so in a known orientation (N or C terminus first). In addition, it is desirable to exert a known force pulling on the peptide, independent of its particular charge. By means of protein expression in cells, Nivia et al.3 have engineered proteins by fusing negatively charged peptides into their C-terminus in order to drag the protein into the nanopore, regardless of the intrinsic charge on the polymer. This procedure will not work on the naturally occurring proteins that one would wish to sequence with a nanopore. Accordingly, this disclosure provides procedures for attaching charged polymers to N termini of naturally-occurring proteins.
Additionally, the current dominant method for proteomic analysis is mass spectrometry, which typically requires as much as a microgram of protein (corresponding to 0.1 nanomoles of a 10 kD protein), though samples of several mg are required for enrichment to detect low abundance (<5%) modified sites (e.g. phosphorylation). In some RT systems for identifying amino acids (e.g., the '509 publication), sample concentrations of about 10 uM and working volumes of 0.1 ml are required, which correspond to a nanomole of sample (i.e., sample requirements are comparable to mass spectrometry). Since samples are delivered to the tunnel junction by diffusion, the quantification of mixed analytes is complicated by differential diffusion and differential binding in the junction. Accordingly, it is desirable to have a method that delivers samples to the junction in a more deterministic way, and uses lower concentrations of analyte.
At least some of these and other goals may be achieved by at least some embodiments of the present disclosure.